Time lapse shooting apparatus and observation method

ABSTRACT

A certain material irregularly expressed in an observation area is effectively observed. An observing apparatus includes a first observing unit performing a time lapse shooting of a predetermined observation area, a first discriminating unit discriminating whether or not a first material is expressed in the observation area based on an image obtained by the first observing unit, and a second observing unit starting a time lapse shooting relating to a part where the first material is expressed at a timing when the first material is expressed in the observation area, in which a shooting frequency of the time lapse shooting by the second observing unit is higher than a shooting frequency of the time lapse shooting by the first observing unit.

CROSS REFERENCE TO THE RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/899,766, filed Feb. 20, 2018, which is a continuation of Ser. No.13/565,281, filed Aug. 2, 2012, which is a continuation application ofInternational Application PCT/JP2011/000468, filed Jan. 28, 2011,designating the U.S., and claims the benefit of priority from JapanesePatent Application No. 2010-022170, filed on Feb. 3, 2010, the entirecontents of which are incorporated herein by reference.

BACKGROUND 1. Field

The present application relates to an observing apparatus performing atime lapse shooting of an observation object such as a living sample andan observation method.

2. Description of the Related Art

Conventionally, a living sample observing apparatus performing a timelapse shooting while incubating cells in an incubation container isknown in Japanese Unexamined Patent Application Publication No.2004-309719.

On the other hand, in recent years, an art manufacturing,differentiating and inducing iPS cells (Induced Pluripotent Stem cell)from cultured cells differentiated into somatic cells is focused andvarious studies have been done in various fields such as a regenerativemedicine field and a development of new drugs field.

As a representative method manufacturing the iPS cells from the culturedcells, a method in which an exogenous gene called as Yamanaka factors(Sox 2, Klf4, Oct3/4, c-Myc) is introduced into the cultured cells witha vector such as retrovirus and plasmid, and they are incubated forseveral weeks is known. The cells to which the exogenous gene isintroduced are reprogrammed (initialized) and come to have equivalentcharacteristics as ES cells (a characteristic multiplying in a colonystate mode, and having pluripotency) in various points. In the cellsinitialized as stated above, an endogenous gene peculiar to the iPScells such as a Nanog gene is expressed. Besides, the expression of theintroduced exogenous gene is terminated simultaneously with theinitialization of cells caused by a mechanism called as a silencing inthe cell.

In this method, it becomes important to monitor a process from theexpression of the exogenous gene to the termination of the expressionthereof caused by the silencing (1) and a process in which theendogenous gene peculiar to the iPS cells is expressed by theinitialization (2) from points of view of an evaluation, an explanationof function of the iPS cells.

It is possible to visualize these processes (1) and (2), and forexample, when the process (1) is visualized, a fluorescent protein gene(DsRed gene) which is expressed together with the expression of theabove-stated exogenous gene (Sox2, Klf4, Oct 3/4, c-Myc) and silencingthereof occurs together with the silencing of the exogenous gene isintroduced into the cultured cells together with the exogenous gene.Besides, when the process (2) is visualized, the endogenous gene (Nanoggene) in experimental animals is recomposed into a gene accompanied bythe fluorescent protein (Nanog-GFP gene) in advance.

However, manufacturing efficiency of the iPS cells is currently low, andtherefore, the iPS cells are not expressed only at a very small part inthe incubation container. Besides, it is currently impossible to predicta part where the iPS cells are expressed. Accordingly, it is difficultto effectively observe the processes (1) and (2) in which the iPS cellsare initialized with the above-stated living sample observing apparatus.

A proposition of the present application is to provide an observingapparatus and an observation method capable of effectively observe acertain material which is irregularly expressed in an observation area.

SUMMARY

An observing apparatus exemplifying the present embodiment includes afirst observing unit performing a time lapse shooting of a predeterminedobservation area, a first discriminating unit discriminating whether ornot a first material is expressed in the observation area based on animage obtained by the first observing unit, and a second observing unitstarting a time lapse shooting relating to a part where the firstmaterial is expressed at a timing when the first material is expressedin the observation area, in which a shooting frequency of the time lapseshooting by the second observing unit is higher than a shootingfrequency of the time lapse shooting by the first observing unit.

An observation method exemplifying the present embodiment includes afirst observing step performing a time lapse shooting of a predeterminedobservation area, a first discriminating step discriminating whether ornot a first material is expressed in the observation area based on animage obtained at the first observing step, and a second observing stepstarting a time lapse shooting relating to a part where the firstmaterial is expressed at a timing when the first material is expressedin the observation area, in which a shooting frequency of the time lapseshooting by the second observing step is higher than a shootingfrequency of the time lapse shooting by the first observing step.

According to the present application, an observing apparatus and anobservation method capable of effectively observe a certain materialwhich is irregularly expressed in an observation area are enabled.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a front view illustrating an overall configuration of a livingsample observing apparatus.

FIG. 2 is a front view of an observing unit 27.

FIG. 3 is a side view of the observing unit 27.

FIG. 4 is an upper surface view of an incubation container 15 (wellplate).

FIG. 5 is a flowchart of an observation process by a controlling unit31.

FIG. 6 is a timing chart representing an obtaining timing of each imagein the observation process.

FIGS. 7A to 7D are views explaining a relationship between a time changeof fluorescence intensities of plural cell colonies different from oneanother and a detailed observation period T1 and a suspended period T2.

FIG. 8 is a graphic chart representing a relationship between a settingtiming of an unimportant ROI and the number of unimportant ROIs.

FIG. 9 is a graphic chart representing a relationship between atransition timing of an important ROI and the number of important ROIs.

DETAILED DESCRIPTION OF THE EMBODIMENT Embodiment

Hereinafter, a living sample observing apparatus is described as anembodiment of the present invention.

FIG. 1 is a front view illustrating an overall configuration of theliving sample observing apparatus. Note that in FIG. 1, a solid lineillustrates a structure of a portion exposed to an external appearance,and a dotted line illustrates a structure of an internal portion whichis not exposed to the external appearance.

As illustrated in FIG. 1, a living sample observing apparatus 1 includesa first casing 11 housing incubation containers accommodating cells anda second casing 12 making up a controlling apparatus. The first casing11 is used under a state mounted on the second casing 12.

A temperature-controlled room 21 covered with a heat insulating materialis formed inside the first casing 11. This temperature-controlled room21 is communicated with outside by a front opening 23 (front door 22)formed at a front face of the first casing 11 and a carry-in entrance 24formed at a left side surface when it is seen from the front of thefirst casing 11.

For example, a temperature controlling mechanism made up of atemperature adjusting apparatus and so on in which Peltier element isused, a humidity controlling mechanism made up of an atomizer and so onspraying mist, a gas controlling mechanism made up of a gas introducingsection and so on coupled to an external carbon dioxide cylinder, anenvironmental sensor detecting an environment of the incubationcontainer in an internal space (all of them are not illustrated) and soon are provided at the temperature-controlled room 21. Inside of thetemperature-controlled room 21 is thereby sealed to maintain theenvironment of the incubation container, and for example, it is kept ata constant temperature by circulating air, and thereby, it is maintainedat a temperature of 37° C., a humidity of 90%, and a carbon dioxideconcentration of 5%, and so on.

Besides, a stocker 25, a container transfer mechanism 26 and anobserving unit 27 are housed in the temperature-controlled room 21 ofthe first casing 11.

The stocker 25 is divided into up and down parts by plural shelves, andthe incubation container 15 (FIG. 2, FIG. 3) can be horizontally housed.

A transfer arm section supporting a holder and various mechanisms(not-illustrated) to transfer the incubation container 15 are providedat the container transfer mechanism 26. The container transfer mechanism26 is able to transfer the holder supported by the transfer arm sectionin a vertical direction (Z direction) or a horizontal direction (X and Ydirections), and to rotate the holder for 180 degrees centering on a Zaxis.

FIG. 2 and FIG. 3 are views illustrating a configuration of theobserving unit 27. A front view of the observing unit 27 is illustratedin FIG. 2, and a side view of the observing unit 27 is illustrated inFIG. 3.

As illustrated in FIG. 2 and FIG. 3, the observing unit 27 is made up ofa diascopic illuminating section 41, a fluorescence epi-illuminatingsection 42, a sample stage 43 and an observing section 44.

The diascopic illuminating section 41 is formed in an arm stateextending from a side part of the sample stage 43 toward an upperdirection, and thereafter, extending to an upper part of the incubationcontainer 15 placed on the sample stage 43. An LED (Light EmittingDiode) for diascopic illumination 51 and an optical system for diascopicillumination 52 are housed in the diascopic illuminating section 41. TheLED for diascopic illumination 51 emits light at a predeterminedwavelength region. The light from the LED for diascopic illumination 51is irradiated on the incubation container 15 placed on the sample stage43 via the optical system for diascopic illumination 52 from an upperside. Note that an illumination (diascopic illumination) by means of thediascopic illuminating section 41 is desirable to be an illumination(dark-field illumination and so on) suitable for visualizing thetransparent incubation container 15 and a transparent phase substance(cells and so on) existing in the incubation container 15.

The fluorescence epi-illuminating section 42 includes LEDs forfluorescence 53 a, 53 b, 53 c and an optical system for fluorescence 54.The LEDs for fluorescence 53 a, 53 b, 53 c emit lights at differentwavelengths from one another. The lights emitted from the LEDs forfluorescence 53 a, 53 b, 53 c are irradiated on the sample stage 43 viathe optical system for fluorescence 54 from a lower side.

The sample stage 43 is made up of a transparent material, and the lightemitted from the diascopic illuminating section 41 and transmitting theincubation container 15, and fluorescence emitted at the incubationcontainer 15 in accordance with excitation light emitted from thefluorescence epi-illuminating section 42 are seldom interrupted andincident on the observing section 44.

An objective lens 55 condensing the light heading from the incubationcontainer 15 to the observing section 44, a stage 56 transferring theincubation container 15 in the vertical direction or the horizontaldirection and so on are also provided at the sample stage 43.

The objective lens 55 at the sample stage 43 is made up of pluralobjective lenses of which magnifications are different (for example, atwo times objective lens, a four times objective lens, a 10 timesobjective lens, a 20 times objective lens, a 40 times objective lens,and so on), and an observation magnification of the observing unit 27can be switched appropriately.

The observing section 44 is made up of a shooting section 57 and animage processing section 58. The shooting section 57 includes animage-forming optical system and an imaging device such as a CCD (ChargeCoupled Device). The image-forming optical system of the observingsection 44 forms an image (diascopic image) by the light transmittingthe incubation container 15 and an image (fluorescence image) by thefluorescence emitted from the incubation container 15 on an imaging areaof the imaging device.

Here, the optical system for fluorescence 54 is able to switch kinds oflight irradiating the incubation container 15 (illumination methods ofthe observing unit 27) between the illumination by the fluorescenceepi-illuminating section 42 (epi-illumination) and the illumination bythe diascopic illuminating section 41 (diascopic illumination) byinserting/detaching a dichroic mirror disposed at a predeterminedposition inside the optical system for fluorescence 54.

For example, the shooting section 57 is able to obtain a diascopic imageof the incubation container 15 under a state in which the illuminationmethod of the observing unit 27 is set to the diascopic illumination,and only the LED for diascopic illumination 51 is lighted from among theabove-stated four LEDs.

Besides, the shooting section 57 is able to obtain a fluorescence imageof the incubation container 15 under a state in which the illuminationmethod of the observing unit 27 is set to the epi-illumination, and anyone of the LEDs for fluorescence 53 a, 53 b, 53 c is lighted from amongthe above-stated four LEDs.

Besides, a wavelength (an excitation wavelength of the observing unit27) of light irradiating the incubation container 15 is switched whenthe lighting LED changes among the three LEDs for fluorescence 53 a, 53b, 53 c under the state in which the illumination method of theobserving unit 27 is set to the epi-illumination.

Further, a wavelength band (a detection channel of the observing unit27) capable of being incident on the shooting section 57 is switchedwhen a fluorescence filter disposed at a predetermined position in theoptical system for fluorescence 54 is switched under the state in whichthe illumination method of the observing unit 27 is set to theepi-illumination.

The image processing section 58 applies an analog signal processincluding an amplifying process and so on for an image (analog imagesignal) obtained by the shooting section 57, and thereafter, performs anA/D (Analog/Digital) conversion of the analog image signal to obtain adigital image signal. This digital image signal is transmitted to acontrolling unit 31.

Returning to FIG. 1, the controlling unit 31 is also housed in additionto a part of the above-stated observing unit 27 in the second casing 12on which the first casing 11 is placed.

The controlling unit 31 controls operations of each section of theliving sample observing apparatus 1. Specifically, the controlling unit31 performs an adjustment of environmental conditions in thetemperature-controlled room 21, carrying in/out of the incubationcontainer 15 to/from the temperature-controlled room 21, an observationof the living sample in the incubation container 15, a transfer of theincubation container 15 in the temperature-controlled room 21, and so onin accordance with an observation schedule or a direct instruction by anoperation of a user.

Besides, the controlling unit 31 displays necessary information on adisplay panel 32 provided at a front surface and so on of the secondcasing 12, and thereby, it is possible to make the user input theobservation schedule by each incubation container. Note that theinformation input from the user to the controlling unit 31 is performedvia an input device (not-illustrated) such as a keyboard coupled to thecontrolling unit 31.

Further, an observation information storing section 33 is provided inthe controlling unit 31, and the observation information storing section33 sequentially stores the digital image signals supplied from theobserving unit 27. The image data of plural images (hereinafter,referred to just as the “image”) are thereby accumulated at theobservation information storing section 33.

Besides, the controlling unit 31 adds index information such as a number(container number) of the incubation container 15 which is an obtainingoriginal of the image and an obtaining time and date of the image forindividual images to manage the plural images accumulated at theobservation information storing section 33 by each incubation containerand by each time and date. Further the observation information storingsection 33 is able to record a change history and so on of theenvironmental conditions (temperature, humidity, carbon dioxideconcentration and so on) in the temperature-controlled room 21.

An image analyzing section 34 performs an image analyzing process forthe images accumulated at the observation information storing section 33according to need.

An observation controlling section 35 controls the observing unit 27 inaccordance with the observation schedule or the direct instruction bythe user's operation. Besides, the observation controlling section 35appropriately changes control contents of the observing unit 27 based ona result of the image analyzing process by the image analyzing section34.

Note that the controlling unit 31 includes a communication unit(not-illustrated) based on a predetermined wireless or wiredcommunication protocol, and it is possible to perform datatransmission/reception with equipments such as external personalcomputers via a network. Accordingly, the user is able to perform theobservation of the incubation container, a setting change of theobserving unit 27, a setting change of the temperature-controlled room21 from a computer located at a remote location.

Here, in the present embodiment, a well plate as illustrated in FIG. 4is assumed as the incubation container 15. The number of wells of theincubation container 15 is, for example, six pieces, and a diameter ofeach well 15A is approximately 30 mm. Various elements required formanufacturing of the iPS cells, namely, a culture solution, feedercells, cultured cells differentiated into somatic cells (mousefibroblast), a vector to introduce Yamanaka factors into the culturedcells, and so on are housed at proper timings in each well 15A. Inparticular, processes (introduction of a DsRed gene, geneticrecombination from a Nanog gene to a Nanog-GFP gene) to visualize theabove-stated processes (1), (2) are performed for the cultured cells inadvance.

When the objective lens 55 of the observing unit 27 is set to be the twotimes objective lens, a size of a range (a visual field 55A of theobserving unit 27) capable of being imaged by one shot within theincubation container 15 is, for example, 4 mm×4 mm. Accordingly, theimagings for 8×8 shots are performed while moving the visual field 55Aof the observing unit 27 on the incubation container 15, and theobtained images of 8×8=64 pieces are connected (tiling) to observealmost a whole of one well 15A as illustrated by a grid line in FIG. 4.

Besides, when the objective lens 55 of the observing unit 27 is set tobe the 20 times objective lens, a size of a range (the visual field ofthe observing unit 27) capable of being imaged by one shot within theincubation container 15 is, for example, 400 μm×400 μm. This size issuitable to observe one cell colony generated in the incubationcontainer 15 in detail.

Besides, an emission wavelength of the LED for fluorescence 53 a of theobserving unit 27 of the present embodiment is set at an excitationwavelength (558 nm) of the DsRed, and an emission wavelength of the LEDfor fluorescence 53 b is set at an excitation wavelength (488 nm) of theNanog-GFP. In this case, the excitation wavelength of the observing unit27 is able to be switched between the excitation wavelength (558 nm) ofthe DsRed and the excitation wavelength (488 nm) of the Nanog-GFP.

Further, the detection channel of the observing unit 27 is able to beswitched between a fluorescence wavelength band (in a vicinity of 583nm. Hereinafter, referred to as a “red color channel”.) of the DsRed anda fluorescence wavelength band (in a vicinity of 520 nm. Hereinafter,referred to as a “green color channel”.) of the Nanog-GFP.

In this case, it is possible to obtain a fluorescence intensitydistribution of the DsRed (DsRed image) existing in the incubationcontainer 15 when the illumination method of the observing unit 27 isset to the epi-illumination, the excitation wavelength of the observingunit 27 is set at 558 nm, and the detection channel of the observingunit 27 is set at the red color channel.

Besides, it is possible to obtain a fluorescence intensity distributionof the Nanog-GFP (GFP image) existing in the incubation container 15when the illumination method of the observing unit 27 is set to theepi-illumination, the excitation wavelength of the observing unit 27 isset at 488 nm, and the detection channel of the observing unit 27 is setat the green color channel.

FIG. 5 is a flowchart of an observation process by the controlling unit31. Hereinafter, respective steps are sequentially described. Note thatit is assumed that the above-stated incubation container 15 is alreadyhoused in the living sample observing apparatus (the container number isassigned and housed in the stocker 25) at a starting time of the flow.

Step S11: The controlling unit 31 makes the user input the observationschedule of the incubation container 15. Here, there are a time lapseshooting of which interval is long (coarse observation), a time lapseshooting of which interval is short (low-frequency detailed observation)and a time lapse shooting of which interval is further short(high-frequency detailed observation) in the observation process of thepresent embodiment. Accordingly, the controlling unit 31 of this stepmakes the user set each of an interval ΔT1 of the coarse observation, aninterval ΔT2 of the low-frequency detailed observation and an intervalΔT3 of the high-frequency detailed observation. The user sets acombination of the intervals ΔT1, ΔT2, ΔT3 to be a combinationsatisfying a relationship as described below.

-   -   ΔT3<ΔT2<ΔT1    -   ΔT2=n×ΔT3 (where “n” is an integer)    -   ΔT1=m×ΔT2 (where “m” is an integer)

Accordingly, for example, they are set as Δ1=8 h, ΔT2=2 h, ΔT3=1 h, andso on.

Note that a necessary time per one round of each of the coarseobservation, the low-frequency detailed observation, the high-frequencydetailed observation are enough shorter compared to these intervals ΔT1,ΔT2, ΔT3, and therefore, they are regarded as zero in this description.

Besides, the controlling unit 31 makes the user set each of a brightnessthreshold value A1 to detect presence/absence of expression of the DsRedand a brightness threshold value A2 to detect presence/absence ofexpression of the Nanog-GFP. The user sets the brightness thresholdvalue A1 to be low when it is necessary to detect the presence/absenceof the expression of the DsRed with high sensitivity, and sets thebrightness threshold value A2 to be low when it is necessary to detectthe presence/absence of the expression of the Nanot-GFP with highsensitivity (incidentally, a necessity to set the brightness thresholdvalue A2 lower than the brightness threshold value A1 is generallyhigh).

Further, the controlling unit 31 makes the user set a magnificationm_(L) of the objective lens to be used for the coarse observation.Hereinafter, the magnification m_(L) is assumed to be set at “2”.

The controlling unit 31 calculates a shot pattern P_(L) (a movingpattern of the stage 56 and a drive timing of the shooting section 57)necessary for imaging all wells of the incubation container 15 in thecoarse observation in accordance with the set magnification m_(L). Notethat here, the magnification m_(L) is set at “2”, and therefore, a shotpattern imaging one well with 8×8 shots as illustrated in FIG. 4 iscalculated as the shot pattern P_(L).

Besides, the controlling unit 31 makes the user set a magnificationm_(H) of the objective lens and a size S_(ROI) of a ROI to be used forthe low-frequency detailed observation. Note that the ROI is an area tobe an object of the low-frequency detailed observation. Hereinafter, itis assumed that the magnification m_(H) is set at “20”, and the sizeS_(ROI) is set at a size of “300 μm×300 μm” corresponding to one cellcolony.

The controlling unit 31 calculates a shot pattern P_(H) necessary forimaging for one ROI with the low-frequency detailed observation inaccordance with a combination of the set magnification m_(H) and thesize S_(ROI). Note that, here, the magnification m_(H) is set at “20”,and the size S_(ROI) is set at “300 μm×300 μm”, and therefore, a shotpattern imaging one ROI by one shot is calculated as the shot patternP_(H).

Besides, the controlling unit 31 makes the user set a period (detailedobservation period) T1 in which the low-frequency detailed observationand the high-frequency detailed observation are to be continued. Thedetailed observation period T1 is set to be a period necessary from thetime when the DsRed is expressed at the cell colony, then the Nanog-GFPis expressed, and thereafter, to the time when it is verified whether ornot the cell colony becomes the iPS cell colony as illustrated in FIGS.7A to 7C. For example, the detailed observation period T1 is set to be504 h (three weeks).

Further, the controlling unit 31 makes the user set a suspended periodT2 to be provided from the start of the low-frequency detailedobservation to an interruption thereof. The suspended period T2 is setat a period (shorter than the detailed observation period T1) necessaryfrom the time when the DsRed is expressed at the cell colony until it isregarded that the Nanog-GFP is not expressed at the cell colony even ifit is waited further more as illustrated in FIG. 7D. For example thesuspended period T2 is set to be 336 h (two weeks).

Step S12: The controlling unit 31 compares the coarse observationschedule defined by the interval ΔT1 and the current time and date, andthereby, discriminates whether or not a shooting time of the coarseobservation comes. When it is discriminated that it comes, thecontrolling unit 31 transfers to step S13, and when it does not come,transfers to step S16.

Step S13: The observation controlling section 35 of the controlling unit31 issues an instruction for the container transfer mechanism 26 todispose the incubation container 15 at the observing unit 27. Thecontainer transfer mechanism 26 takes out the incubation container 15from the stocker 25, and disposes it at a predetermined position on thestage 56 of the observing unit 27. Note that in the step S13 for asecond time or later, the transfer of the incubation container 15 is notperformed when the incubation container 15 is already disposed at thestage 56.

Subsequently, the observation controlling section 35 of the controllingunit 31 inputs the information of the magnification m_(L) and the shotpattern P_(L), and the shooting instruction of the coarse observation tothe observing unit 27. The observing unit 27 receiving the above obtainstwo kinds of well images I_(P), I_(R) as for all of the wells by thefollowing procedures (a) to (d).

(a) The observing unit 27 sets the magnification of the objective lensat the magnification m_(L) (here, two times), and sets the illuminationmethod to the diascopic illumination.

(b) The observing unit 27 drives the stage 56 and the shooting section57 with the shot pattern P_(L) while lighting only the LED for diascopicillumination 51 from among the above-stated four LEDs, and thereby,obtains the diascopic images (here, the diascopic images of 8×8×6pieces) relating to all of the wells in the incubation container 15.These diascopic images are stored at the observation information storingsection 33 of the controlling unit 31.

(c) The observing unit 27 switches the illumination method into theepi-illumination.

(d) The observing unit 27 drives the stage 56 and the shooting section57 with the shot pattern P_(L) while lighting the LED for fluorescence53 a under a state in which the detection channel is set at the redcolor channel, and thereby, obtains the DsRed images (here, the DsRedimages of 8×8×6 pieces) relating to all of the wells in the incubationcontainer 15. These DsRed images are stored at the observationinformation storing section 33 of the controlling unit 31.

Subsequently, the image analyzing section 34 of the controlling unit 31creates the diascopic images for each well (six pieces of well imagesI_(P)) by connecting the diascopic images obtained from the same wellwith each other in an image obtaining order from among the diascopicimages (here the 8×8×6 pieces of diascopic images) obtained by theprocedure (b). Each of the above-stated six pieces of well images I_(P)is stored at the observation information storing section 33 togetherwith a number of the well (well number) which is the obtaining originalof each image and the obtaining time and date of each image. Besides,the image analyzing section 34 of the controlling unit 31 creates theDsRed images for each well (six pieces of well images I_(R)) byconnecting the DsRed images obtained from the same well with each otherin the image obtaining order from among the DsRed images (here the 8×8×6pieces of DsRed images) obtained by the procedure (d). Each of theabove-stated six pieces of well images I_(R) is stored at theobservation information storing section 33 together with the well numberof the well which is the obtaining original of each image and theobtaining time and date of each image.

Step S14: The image analyzing section 34 of the controlling unit 31compares each of the well images I_(R) (six pieces of well images I_(R))obtained at the last step S13 with the brightness threshold value A1,and searches one or plural high-brightness area(s) which exceed(s) thebrightness threshold value A1 among the six pieces of well images I_(R)as a setting destination of the ROI. The controlling unit 31 transfersto step S15 if even one high-brightness area is detected, and thecontrolling unit 31 transfers to step S16 if no high-brightness area isdetected. Note that in the step S14 for a second time or later, the areawhere the ROI (or a later-described important ROI) is already set at theformer step S14 is excluded from the search range among the six piecesof well images I_(R).

Step S15: The image analyzing section 34 of the controlling unit 31 setsthe ROI for each of the one or plural high-brightness area(s) detectedat the step S14 by the following procedures (e) to (g).

(e) The image analyzing section 34 calculates a coordinate correspondingto a center of the high-brightness area (or a gravity center ofbrightness) on the well image I_(R) to be the detection original of thehigh-brightness area.

(f) The image analyzing section 34 calculates a coordinate correspondingto the center of the high-brightness area on the incubation container 15based on the coordinate calculated at the procedure (e) and the wellnumber of the well image I_(R).

(g) The image analyzing section 34 sets the ROI for the area with thesize S_(ROI) centering on the coordinate calculated at the procedure(f).

The image analyzing section 34 of the controlling unit 31 suppliesinformation of a setting time and date for each of one or plural ROIsset on the incubation container 15, and supplies a number (ROI number)for each of the ROIs in a setting order. Note that the supply of the ROInumber is performed not by each incubation container 15 but by each wellof the incubation container 15, and the ROI number supplied for thefirst ROI of a certain well in the step S15 for a second time or lateris the number subsequent to the ROI number supplied for the last ROI ofthe same well at the former step S15.

Step S16: The controlling unit 31 discriminates whether or not ashooting time of the low-frequency detailed observation comes bycomparing a schedule of the low-frequency detailed observation definedby the interval ΔT2 and the current time and date, then transfers tostep S17 when it comes, and transfers to step S23 when it does not come.

Step S17: The controlling unit 31 discriminates whether or not even oneROI is set at the incubation container 15, and transfers to step S18when the ROI is set, and transfers to the step S23 when it is not set.

Step S18: The observation controlling section 35 of the controlling unit31 issues an instruction for the container transfer mechanism 26 todispose the incubation container 15 at the observing unit 27. Thecontainer transfer mechanism 26 takes out the incubation container 15from the stocker 25, and disposes at the predetermined position on thestage 56 of the observing unit 27. Note that the transfer of theincubation container 15 is not performed when the incubation container15 is already disposed on the stage 56.

The observation controlling section 35 of the controlling unit 31 inputsthe information of the magnification m_(H) and the shot pattern P_(H),coordinate information of the set ROI and a shooting instruction of thelow-frequency detailed observation to the observing unit 27. Theobserving unit 27 receiving the above obtains three kinds of ROI imagesI_(P)′, I_(R)′, I_(G)′ as for all of the set ROIs by the followingprocedures (a′) to (k′).

(a′) The observing unit 27 sets the magnification of the objective lensat the magnification m_(H) (here, 20 times), and sets the illuminationmethod to the diascopic illumination.

(b′) The observing unit 27 sets the coordinate of the stage 56 so that acenter of the ROI positioning at an endmost among the set ROIs is on anoptical axis of the objective lens 55.

(c′) The observing unit 27 drives the stage 56 and the shooting section57 with the shot pattern P_(H) while lighting only the LED for diascopicillumination 51 from among the above-stated four LEDs, and thereby, thediascopic image (here, one piece of diascopic image) relating to the ROIis obtained. This diascopic image is stored at the observationinformation storing section 33 of the controlling unit 31.

(d′) The observing unit 27 repeats the procedure (c′) while changing theROI disposed on the optical axis, and thereby, obtains the diascopicimages relating to all of the ROIs.

(e′) The observing unit 27 switches the illumination method into theepi-illumination.

(f′) The observing unit 27 sets the coordinate of the stage 56 so thatthe center of the ROI positioning at the endmost among the set ROIs ison the optical axis of the objective lens 55.

(g′) The observing unit 27 drives the stage 56 and the shooting section57 with the shot pattern P_(H) while lighting the LED for fluorescence53 a under a state in which the detection channel is set at the redcolor channel, and thereby, obtains the DsRed image (here, one piece ofDsRed image) relating to the ROI. This DsRed image is stored at theobservation information storing section 33 of the controlling unit 31.

(h′) The observing unit 27 repeats the procedure (g′) while changing theROI disposed on the optical axis, and thereby, obtains the DsRed imagesrelating to all of the ROIs.

(i′) The observing unit 27 sets the coordinate of the stage 56 so thatthe center of the ROI positioning at the endmost among the set ROIs ison the optical axis of the objective lens 55.

(j′) The observing unit 27 drives the stage 56 and the shooting section57 with the shot pattern P_(H) while lighting the LED for fluorescence53 b under a state in which the detection channel is set at the greencolor channel, and thereby, obtains the GFP image (here, one piece ofGFP image) relating to the ROI. This GFP image is stored at theobservation information storing section 33 of the controlling unit 31.

(k′) The observing unit 27 repeats the procedure (j′) while changing theROI disposed on the optical axis, and thereby, obtains the GFP imagesrelating to all of the ROIs.

Subsequently, the image analyzing section 34 of the controlling unit 31creates the diascopic image (ROI image I_(P)′) for each ROI byconnecting the images obtained from the same ROI with each other in theimage obtaining order from among the diascopic images obtained by theprocedures (b′) to (d′) (note that here, the number of diascopic imagerelating to one ROI is one piece, and therefore, the diascopic image isset to be the ROI image I_(P)′ as it is without performing theconnection). Each of the created ROI images I_(P)′ is stored at theobservation information storing section 33 together with the ROI numberof the ROI which is the obtaining original of each image and theobtaining time and date of each image.

Besides, the image analyzing section 34 of the controlling unit 31creates the DsRed image (ROI image I_(R)′) for each ROI by connectingthe images obtained from the same ROI with each other in the imageobtaining order from among the DsRed images obtained by the procedures(f′) to (h′) (note that here, the number of DsRed image relating to oneROI is one piece, and therefore, the DsRed image is set to be the ROIimage I_(R)′ as it is without performing the connection). Each of thecreated ROI images I_(R)′ is stored at the observation informationstoring section 33 together with the ROI number of the ROI which is theobtaining original of each image and the obtaining time and date of eachimage.

Besides, the image analyzing section 34 of the controlling unit 31creates the GFP image (ROI image I_(G)′) for each ROI by connecting theimages obtained from the same ROI with each other in the image obtainingorder from among the GFP images obtained by the procedures (i′) to (k′)(note that here, the number of GFP image relating to one ROI is onepiece, and therefore, the GFP image is set to be the ROI image I_(G)′ asit is without performing the connection). Each of the created ROI imagesI_(G)′ is stored at the observation information storing section 33together with the ROI number of the ROI which is the obtaining originalof each image and the obtaining time and date of each image.

Step S19: The image analyzing section 34 of the controlling unit 31compares a brightness average value of one or plural ROI image(s) I_(G)′obtained at the last step S18 with the brightness threshold value A2,and thereby, finds the one which is brighter than the brightnessthreshold value A2 (high-brightness ROI) from among the set ROIs. Wheneven one high-brightness ROI is found, the controlling unit 31 transfersto step S20, and the controlling unit 31 transfers to the step S23 whenno high-brightness ROI is found.

Step S20: The image analyzing section 34 of the controlling unit 31transfers the one or plural high-brightness ROI(s) found at the step S19to important ROI(s). Note that the important ROI is an area to be anobject of the high-frequency detailed observation (hereinafter, a mereROI is referred to as an “unimportant ROI” to distinguish it from theimportant ROI). Besides, the image analyzing section 34 suppliesinformation of a transferred time and date for the important ROI whichis transferred from the unimportant ROI to the important ROI in thisstep.

Step S21: The image analyzing section 34 of the controlling unit 31compares each of setting times and dates (the time and date when the ROIis first set to be the unimportant ROI) of all of the unimportant ROIswith the current time and date, and thereby, finds the unimportant ROIin which the suspended period T2 elapses since the setting time and date(expired ROI). When even one expired ROI is found, the controlling unit31 transfers to step S22, and the controlling unit 31 transfers to thestep S23 when no expired ROI is found.

Step S22: The image analyzing section 34 of the controlling unit 31eliminates one or plural expired ROI(s) found at the step S21.

Step S23: The controlling unit 31 compares a schedule of thehigh-frequency detailed observation defined by the interval ΔT3 and thecurrent time and date to thereby discriminate whether or not a shootingtime of the high-frequency detailed observation comes. The controllingunit 31 transfers to step S24 when it comes and transfers to step S28when it does not come.

Step S24: The controlling unit 31 discriminates whether or not even oneimportant ROI is set at the incubation container 15. The controllingunit 31 transfers to step S25 when it is set, and transfers to the stepS28 when it is not set.

Step S25: The observation controlling section 35 of the controlling unit31 issues an instruction for the container transfer mechanism 26 todispose the incubation container 15 at the observing unit 27. Thecontainer transfer mechanism 26 takes out the incubation container 15from the stocker 25, and disposes at the predetermined position on thestage 56 of the observing unit 27. Note that when the incubationcontainer 15 is already disposed at the stage 56, the transfer of theincubation container 15 is not performed.

The observation controlling section 35 of the controlling unit 31 inputsthe information of the magnification m_(H) and the shot pattern P_(H),coordinate information of a set important ROI, and the shootinginstruction of the high-frequency detailed observation to the observingunit 27. The observing unit 27 receiving the above obtains three kindsof ROI images I_(P)′, I_(R)′, I_(G)′ as for all of the set importantROIs by the following procedures (a″) to (k″).

(a″) The observing unit 27 sets the magnification of the objective lensat the magnification m_(H) (here, 20 times), and sets the illuminationmethod to the diascopic illumination.

(b″) The observing unit 27 sets the coordinate of the stage 56 so that acenter of the important ROI positioning at an endmost among the setimportant ROIs is on the optical axis of the objective lens 55.

(c″) The observing unit 27 drives the stage 56 and the shooting section57 with the shot pattern P_(H) while lighting only the LED for diascopicillumination 51 from among the above-stated four LEDs, and thereby,obtains the diascopic image (here, one piece of diascopic image)relating to the important ROI. This diascopic image is stored at theobservation information storing section 33 of the controlling unit 31.

(d″) The observing unit 27 repeats the procedure (c″) while changing theimportant ROI disposed on the optical axis, and thereby, obtains thediascopic images relating to all of the important ROIs.

(e″) The observing unit 27 switches the illumination method to theepi-illumination.

(f″) The observing unit 27 sets the coordinate of the stage 56 so thatthe center of the important ROI positioning at the endmost among the setimportant ROIs is on the optical axis of the objective lens 55.

(g″) The observing unit 27 drives the stage 56 and the shooting section57 with the shot pattern P_(H) while lighting the LED for fluorescence53 a under a state in which the detection channel is set at the redcolor channel, and thereby, obtains the DsRed image (here, one piece ofDsRed image) relating to the important ROI. This DsRed image is storedat the observation information storing section 33 of the controllingunit 31.

(h″) The observing unit 27 repeats the procedure (g″) while changing theimportant ROI disposed on the optical axis, and thereby, obtains theDsRed images relating to all of the important ROIs

(i″) The observing unit 27 sets the coordinate of the stage 56 so thatthe center of the important ROI positioning at the endmost among the setimportant ROIs is on the optical axis of the objective lens 55.

(j″) The observing unit 27 drives the stage 56 and the shooting section57 with the shot pattern P_(H) while lighting the LED for fluorescence53 b under a state in which the detection channel is set at the greencolor channel, and thereby, obtains the GFP image (here, one piece ofGFP image) relating to the important ROI. This GFP image is stored atthe observation information storing section 33 of the controlling unit31.

(k″) The observing unit 27 repeats the procedure (j″) while changing theimportant ROI disposed on the optical axis, and thereby, obtains the GFPimages relating to all of the important ROIs.

Subsequently, the image analyzing section 34 of the controlling unit 31creates the diascopic image (ROI image I_(P)′) for each important ROI byconnecting the images obtained from the same important ROI with eachother in the image obtaining order from among the diascopic imagesobtained by the procedures (b″) to (d″) (note that here, the number ofdiascopic images relating to one important ROI is one piece, andtherefore, the diascopic image is set to be the ROI image I_(P)′ as itis without performing the connection). Each of the created ROI imagesI_(P)′ is stored at the observation information storing section 33together with the ROI number of the important ROI which is the obtainingoriginal of each image and the obtaining time and date of each image.

Besides, the image analyzing section 34 of the controlling unit 31creates the DsRed image (ROI image I_(R)′) for each important ROI byconnecting the images obtained from the same important ROI in the imageobtaining order from among the DsRed images obtained by the procedures(f″) to (h″) (note that here, the number of DsRed images relating to oneimportant ROI is one piece, and therefore, the DsRed image is set to bethe ROI image I_(R)′ as it is without performing the connection). Eachof the above-stated ROI images I_(R)′ is stored at the observationinformation storing section 33 together with the ROI number of theimportant ROI which is the obtaining original of each image and theobtaining time and date of each image.

Besides, the image analyzing section 34 of the controlling unit 31creates the GFP image (ROI image I_(G)′) for each important ROI byconnecting the images obtained from the same important ROI with eachother in the image obtaining order from among the GFP images obtained bythe procedures (i″) to (k″) (note that here, the number of GFP imagesrelating to one important ROI is one piece, and therefore, the GFP imageis set to be the ROI image I_(G)′ as it is without performing theconnection). Each of the created ROI images I_(G)′ is stored at theobservation information storing section 33 together with the ROI numberof the important ROI which is the obtaining original of each image andthe obtaining time and date of each image.

Step S26: The image analyzing section 34 of the controlling unit 31compares each of the set dates and hours of all of the important ROIs(the time and date when the ROI is first set to be the important ROI)with the current time and date, and thereby, finds the important ROI inwhich an elapsed time since the setting time and date exceeds thedetailed observation period T1 elapses (already observed ROI). When evenone already observed ROI is found, the controlling unit 31 transfers tostep S27, and the controlling unit 31 transfers to the step S28 when noalready observed ROI is found.

Step S27: The image analyzing section 34 of the controlling unit 31eliminates one or plural already observed ROI(s) found at the step S26.

Step S28: The controlling unit 31 issues an instruction for thecontainer transfer mechanism 26 to house the incubation container 15into the stocker 25. The container transfer mechanism 26 takes out theincubation container 15 from the observing unit 27, and houses it at apredetermined position of the stocker 25. Note that the transfer of theincubation container 15 is not performed when the incubation container15 is already housed in the stocker 25 at that time.

Step S29: The controlling unit 31 discriminates whether or not a certainperiod (suspended period T3) elapses since any one of the important ROIsand the unimportant ROIs is not set on the incubation container 15. Whenthe certain period elapses, the controlling unit 31 finishes the flow,and returns to the step S12 if it does not elapse. Note that when theunimportant ROI has never set on the incubation container 15, thecontrolling unit 31 does not perform the discrimination, and returns tothe step S12 (hereinabove is the description of the flow).

FIG. 6 is a timing chart representing an obtaining timing of each imagein the above-stated observation process. Note that only an image of acertain well of the incubation container 15 is focused for easy tounderstanding in FIG. 6.

When the observation process is started, at first, the coarseobservation is started as represented at an upper part in FIG. 6. Theimages obtained in respective rounds of the coarse observation are twokinds of the well images I_(P), I_(R), and the interval of the coarseobservation is the relatively long interval ΔT1.

After that, when a high-brightness area exceeding the brightnessthreshold value A1 is expressed in the well image I_(R) obtained by thecoarse observation, the unimportant ROI (first unimportant ROI) is setat the area, as illustrated in FIG. 6(A).

Subsequently, when another high-brightness area exceeding the brightnessthreshold value A1 is expressed in the well image I_(R) obtained by thecoarse observation, the unimportant ROI (second unimportant ROI) is setat the area, as illustrated in FIG. 6(B).

Besides, when the first unimportant ROI is set (FIG. 6(A)), thelow-frequency detailed observation relating to the first unimportant ROIis started as illustrated in FIG. 6(A′).

Further, when the second unimportant ROI is set (FIG. 6(B)), thelow-frequency detailed observation relating to the second unimportantROI is started as illustrated in FIG. 6(B′).

The images obtained by respective rounds of the low-frequencyobservation are three kinds of ROI images I_(P), I_(R), I_(G), and theinterval of the low-frequency detailed observation is the short intervalΔT2.

When an average brightness of the ROI image I_(G)′ obtained by thelow-frequency detailed observation of the first unimportant ROI exceedsthe brightness threshold value A2, the first unimportant ROI transfersto the important ROI as illustrated in FIG. 6(C).

Besides, when an average brightness of the ROI image I_(G)′ obtained bythe low-frequency detailed observation of the second unimportant ROIexceeds the brightness threshold value A2, the second unimportant ROItransfers to the important ROI as illustrated in FIG. 6(D).

When the first unimportant ROI transfers to the important ROI (FIG.6(C)), the high-frequency detailed observation relating to the importantROI is started.

Besides, when the second unimportant ROI transfers to the important ROI(FIG. 6(D)), the high-frequency detailed observation relating to theimportant ROI is started.

The images obtained by respective rounds of the high-frequencyobservation are three kinds of ROI images I_(P), I_(R), I_(G), and theinterval of the high-frequency detailed observation is the further shortinterval ΔT3.

Namely, the living sample observing apparatus according to the presentembodiment performs the coarse observation of the whole well, and thenstarts the low-frequency detailed observation of a cell colony at thetime when the cell colony having high possibility to be the iPS cellcolony is generated in the well. The low-frequency detailed observationis transferred to the high-frequency detailed observation at the timewhen the possibility in which the cell colony being the object of thelow-frequency detailed observation becomes the iPS cell colony isfurther increased.

FIGS. 7A to 7D are views representing time changes of fluorescenceintensities of four cell colonies being the objects of the detailedobservation in the above-stated observation process. Note that in FIGS.7A to 7D, a reference symbol “R” represents the fluorescence intensityof the DsRed, and a reference symbol “G” represents the fluorescenceintensity of the Nanog-GFP.

The time change represented in FIG. 7A relates to the cell colony inwhich the silencing occurs after the DsRed is expressed, and theNanog-GFP is expressed to be the iPS cell colony before the expressionof the DsRed completely terminates.

Besides, the time change represented in FIG. 7B relates to the cellcolony in which the silencing occurs after the DsRed is expressed, andthe Nanog-GFP is expressed to be the iPS cell colony after theexpression of the DsRed terminates.

Besides, the time change represented in FIG. 7C relates to the cellcolony in which the silencing occurs after the DsRed is expressed, andthe Nanog-GFP is expressed after the expression of the DsRed terminates,but after that, the cell colony does not become the iPS cell colonybecause the expression of the Nanog-GFP terminates for some reason.

Further, the time change represented in FIG. 7D relates to the cellcolony in which the silencing occurs after the DsRed is expressed, butthe cell colony does not become the iPS cell colony because theNanog-GFP is not expressed for some reason.

Among the above, the low-frequency detailed observation is started atthe time when the fluorescence intensity of the DsRed exceeds thebrightness threshold value A1, transfers to the high-frequency detailedobservation when the fluorescence intensity of the Nanog-GFP exceeds thebrightness threshold value A2, and the detailed observation finishes atthe time when the detailed observation period T1 elapses since the starttime of the low-frequency detailed observation as for the cell coloniesin which both of the DsRed and the Nanog-GFP are expressed (FIGS. 7A to7C).

Accordingly, the time change of the fluorescence intensity of the DsRedand the time change of the fluorescence intensity of the Nanog-GFP areeach imaged at a proper frequency and for a proper period as for thecell colonies in which both of the DsRed and the Nanog-GFP are expressed(FIGS. 7A to 7C).

On the other hand, the low-frequency detailed observation is started atthe time when the fluorescence intensity of the DsRed exceeds thebrightness threshold value A1, and the detailed observation isinterrupted at the time when the suspended period T2 elapses since thestart time thereof as for the cell colony in which only the former oneis expressed between the DsRed and the Nanog-GFP (FIG. 7D).

Accordingly, only the time change of the fluorescence intensity of theDsRed is imaged at the proper frequency and for the proper period as forthe cell colony in which only the former one is expressed between theDsRed and the Nanog-GFP (FIG. 7D).

Namely, the living sample observing apparatus according to the presentembodiment images respective states of various cell colonies each at theproper frequency and for the proper period.

As a result, the living sample observing apparatus according to thepresent embodiment is able to effectively observe the iPS cell colonyirregularly expressed at the incubation container 15.

Here, it is necessary to irradiate excitation light to perform thefluorescence observation of the cells at the incubation container 15,and an irradiation amount of the excitation light is to be suppressed asmuch as possible because the fluorescence observation involves damageson the cells. Accordingly, the present embodiment in which the timing,the frequency, and so on observing a narrow range of the incubationcontainer 15 in detail (perform a micro observation) are appropriatelyset in accordance with the states of the cells is effective to suppressthe damages on the cells.

Further, there is a possibility in which self-fluorescence from the cellcolony disturbs the observation of an object colony when a wide range ofthe incubation container 15 is coarsely observed (perform a macroobservation), and therefore, the present embodiment appropriatelyperforming the micro observation is effective.

[Supplementary Items of the Embodiments]

Note that the controlling unit 31 of the above-stated embodiment maycreate, for each well or for each incubation container, a histogramrepresenting a relationship between the setting timing of theunimportant ROI and the number of set unimportant ROIs as, for example,represented in FIG. 8, and stores the histogram to the observationinformation storing section 33 after the observation process illustratedin FIG. 5 finishes or during the execution process of the observationprocess. This histogram is a histogram of the expression timing of theDsRed.

Besides, the controlling unit 31 of the above-stated embodiment maycreate, for each well or for each incubation container, a histogramrepresenting a relationship between a transfer timing of the importantROI (the transfer timing from the unimportant ROI to the important ROI)and the number of important ROIs as, for example, represented in FIG. 9,and stores the histogram to the observation information storing section33 after the observation process illustrated in FIG. 5 finishes orduring the execution process of the observation process. This histogramis a histogram of the expression timing of the Nanog-GFP.

Further, the controlling unit 31 of the above-stated embodiment maycreate a time lapse moving image (a time lapse moving image of theDsRed) by each ROI number by connecting the plural ROI images I_(R) (theROI image I_(R) of the unimportant ROI and the ROI image I_(R) of theimportant ROI) of which ROI number is in common in the image obtainingorder, and stores the image to the observation information storingsection 33 after the observation process illustrated in FIG. 5 finishesor during the execution process of the observation process. Note thatthe interval of the ROI image I_(R) of the unimportant ROI if wider thanthat of the ROI image I_(R) of the important ROI, and therefore, it isdesirable to interpolate the ROI image I_(R) of the unimportant ROI in atime direction when both of them are connected.

Similarly, the controlling unit 31 of the above-stated embodiment maycreate a time lapse moving image (a time lapse moving image of theNanog-GFP) by each ROI number by connecting the plural ROI images I_(G)(the ROI image I_(G) of the unimportant ROI and the ROI image I_(G) ofthe important ROI) of which ROI number is in common in the imageobtaining order, and stores the image to the observation informationstoring section 33 after the observation process illustrated in FIG. 5finishes or during the execution process of the observation process.Note that the interval of the ROI image I_(G) of the unimportant ROI ifwider than that of the ROI image I_(G) of the important ROI, andtherefore, it is desirable to interpolate the ROI image I_(G) of theunimportant ROI in the time direction when both of them are connected.

Similarly, the controlling unit 31 of the above-stated embodiment maycreate a time lapse moving image (a time lapse moving image of thediascopic image) by each ROI number by connecting the plural ROI imagesI_(P) (the ROI image IP of the unimportant ROI and the ROI image I_(P)of the important ROI) of which ROI number is in common in the imageobtaining order, and stores the image to the observation informationstoring section 33 after the observation process illustrated in FIG. 5finishes or during the execution process of the observation process.Note that the interval of the ROI image I_(P) of the unimportant ROI ifwider than that of the ROI image I_(P) of the important ROI, andtherefore, it is desirable to interpolate the ROI image I_(P) of theunimportant ROI in the time direction when both of them are connected.

Further, the controlling unit 31 of the above-stated embodiment mayautomatically discriminate whether or not the cell colony taken in thetime lapse moving images is the iPS cell colony based on the DsRed timelapse moving image and the Nanog-GFP time lapse moving image of whichROI number is in common.

For example, the controlling unit 31 may compare a latest frame of theDsRed time lapse moving image and a latest frame of the Nanog-GFP timelapse moving image, and regards that the cell colonies taken in the timelapse moving images are the iPS cell colonies when the latter one islarger than the former one for a certain degree or more, and otherwise,regards that the cell colonies taken in the time lapse moving images arenon-iPS cell colonies.

According to the discrimination method as stated above, it is possibleto determine that the cell colonies going through the fluorescenceintensity change as illustrated in FIGS. 7A and 7B are the iPS cellcolonies, and the cell colonies going through the fluorescence intensitychange as illustrated in FIGS. 7C and 7D are the non-iPS cell colonies.

Besides, in the above-stated embodiment, the incubation container 15 isassumed to be the well plate, but it may be the other incubationcontainers such as a dish and a flask.

The many features and advantages of the embodiment are apparent from thedetailed specification and, thus, it is intended by the appended claimsto cover all such features and advantages of the embodiment that fallwithin the true spirit and scope thereof. Further, since numerousmodifications and changes will readily occur to those skilled in theart, it is not desired to limit the inventive embodiment to the exactconstruction and operation illustrated and described, and accordinglyall suitable modifications and equivalents may be resorted to, fallingwithin the scope thereof.

What is claimed is:
 1. An observing apparatus, comprising: animage-forming optical system; an input device configured to receive aninput from a user; an imaging device; and a controller configured to:connect to the input device, control an illumination device and theimaging device to perform a first time lapse shooting of a firstobservation area at a first shooting frequency, determine whether or nota first material is expressed in the first observation area based on afirst threshold set via the input device and an image obtained duringthe first time lapse shooting, and control the illumination device andthe image device to perform a second time lapse shooting of a secondobservation area at a timing when the first material is expressed in thefirst observation area and at a second shooting frequency that is higherthan the first shooting frequency.
 2. The observing apparatus accordingto claim 1, wherein the controller is further configured to: determinewhether or not a second material different from the first material isexpressed in the second observation area based on a second threshold setvia the input device and an image obtained by the imaging device, andcontrol the illumination device and the imaging device to increase thesecond shooting frequency of the second time lapse shooting.
 3. Theobserving apparatus according to claim 1, wherein the input device isconfigured to set the first time lapse shooting and the second timelapse shooting to the second shooting frequency that is higher than thefirst shooting frequency.
 4. The observing apparatus according to claim2, wherein the controller is further configured to interrupt the secondtime lapse shooting when the second material is not expressed after apredetermined time elapses since the first material is expressed.
 5. Theobserving apparatus according to claim 2, wherein the input device isconfigured to set the first time lapse shooting and the second timelapse shooting, both of a first image to observe the first material anda second image to observe the second material are obtained in the secondtime lapse shooting, and the first image is obtained in the first timelapse shooting.
 6. The observing apparatus according to claim 2, whereinthe controller is further configured to start the second time lapseshooting relating to each of the plurality of parts at each expressiontiming when the first material is expressed at a plurality of partsdifferent from one another in the first observation area at differenttimings.
 7. The observing apparatus according to claim 6, furthercomprising a statistics obtaining unit which finds a relationshipbetween an expression timing of the first material and the number ofparts where the first material is expressed.
 8. The observing apparatusaccording to claim 6, further comprising a statistics obtaining unitconfigured to determine a relationship between an expression timing ofthe second material and the number of parts where the second material isexpressed.
 9. The observing apparatus according to claim 2, furthercomprising: a laser light source configured to irradiate laser light onthe first observation area; and an imaging section configured to detectfluorescence, wherein the controller is further configured to: compare alight intensity of fluorescence of the first material being detected bythe imaging section with a predetermined level set via the input deviceto determine whether or not the first material is expressed, and comparea light intensity of fluorescence of the second material being detectedby the imaging section with a predetermined level set via the inputdevice to determine whether or not the second material is expressed. 10.The observing apparatus according to claim 1, wherein the firstobservation area is all or a part of an area of an incubation containerincubating a living sample, and the observing apparatus furthercomprises an environmental controlling unit configured to control anenvironment of the incubation container.
 11. An observation method,comprising: receiving input from a user via an input device; performinga first time lapse shooting of a first observation area at a firstshooting frequency; determining whether or not a first material isexpressed in the first observation area based on a first threshold setvia the input device and an image obtained during the first time lapseshooting; and performing a second time lapse shooting of a secondobservation area at a timing when the first material is expressed in thefirst observation area and at a second shooting frequency that is higherthan the first shooting frequency.
 12. The observation method accordingto claim 11, further comprising: determining whether or not a secondmaterial different from the first material is expressed in the secondobservation area based on a second threshold set via the input deviceand an image obtained by the imaging device; and increasing the secondshooting frequency of the second time lapse shooting.
 13. Theobservation method according to claim 12, further comprising:irradiating laser light at the first observation area; and detectingfluorescence, wherein determining whether or not the first material isexpressed in the first observation area by comparing light intensity offluorescence of the first material with a predetermined level set viathe input device, and determining whether or not the second material isexpressed by comparing light intensity of fluorescence of the secondmaterial with a predetermined level set via the input device.
 14. Theobservation apparatus according to claim 1, wherein the input from theuser comprising one or more of an interval time, a brightness thresholdfor ROI identification, an ROI size, and an objective lensmagnification.
 15. The observation method according to claim 11, whereinthe input from the user comprising one or more of an interval time, abrightness threshold for ROI identification, an ROI size, and anobjective lens magnification.